Nanopore sequencing for the detection and identification of <i>Xylella fastidiosa</i> subspecies and sequence types from naturally infected plant material

Faino, Luigi; Scala, Valeria; Albanese, Alessio; Modesti, Vanessa; Grottoli, Alessandro; Pucci, Nicoletta; Doddi, Andrea; L’Aurora, Alessia; Tatulli, Giuseppe; Reverberi, Massimo; Loreti, Stefania

doi:10.1111/ppa.13416

Cited by 19 publications

(21 citation statements)

References 43 publications

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“…However, when the MinION was used previously to detect X. fastidiosa , the obtained sequencing reads were simply aligned to reference genomes to find the genome to which the highest number of reads mapped without further phylogenetic analysis [17]; identification was limited to subspecies, or sequencing was performed only after PCR of MLST loci making whole-genome-based phylogenetic analysis impossible [45]. More recently, the Illumina iSeq and MiSeq were used for detection and identification of X.…”

Section: Discussionmentioning

confidence: 99%

Investigating plant disease outbreaks with long-read metagenomics: sensitive detection and highly resolved phylogenetic reconstruction applied to Xylella fastidiosa

et al. 2022

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Early disease detection is a prerequisite for enacting effective interventions for disease control. Strains of the bacterial plant pathogen Xylella fastidiosa have recurrently spread to new crops in new countries causing devastating outbreaks. So far, investigation of outbreak strains and highly resolved phylogenetic reconstruction have required whole-genome sequencing of pure bacterial cultures, which are challenging to obtain due to the fastidious nature of X. fastidiosa . Here, we show that culture-independent metagenomic sequencing, using the Oxford Nanopore Technologies MinION long-read sequencer, can sensitively and specifically detect the causative agent of Pierce’s disease of grapevine, X. fastidiosa subspecies fastidiosa . Using a DNA sample from a grapevine in Virginia, USA, it was possible to obtain a metagenome-assembled genome (MAG) of sufficient quality for phylogenetic reconstruction with SNP resolution. The analysis placed the MAG in a clade with isolates from Georgia, USA, suggesting introduction of X. fastidiosa subspecies fastidiosa to Virginia from the south-eastern USA. This proof of concept study, thus, revealed that metagenomic sequencing can replace culture-dependent genome sequencing for reconstructing transmission routes of bacterial plant pathogens.

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Section: Discussionmentioning

confidence: 99%

Investigating plant disease outbreaks with long-read metagenomics: sensitive detection and highly resolved phylogenetic reconstruction applied to Xylella fastidiosa

et al. 2022

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“…Metagenomics, the study of genetic material from environmental samples, beyond whole-genome sequencing, allows for the detection of strains from several subspecies and ST at the same time from the host ( 20 ). Recently, the use of long-read sequencing as diagnostic tool identified Xf subspecies and ST from infected samples ( 12 , 21 ).…”

Section: Introductionmentioning

confidence: 99%

Metagenomic Sequencing for Identification of Xylella fastidiosa from Leaf Samples

et al. 2021

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“…The effective management of quarantine pathogens according to international regulations requires fast, sensitive, and cost-effective diagnostic tests that not only detect the pathogen but also distinguish specific genotypes in a quantitative manner [27,37,38]. Cur- 2) correlated with the quantity of XF detected by qPCR (Ct method) in the same samples.…”

Section: Discussionmentioning

confidence: 99%

“…The Oxford Nanopore Technologies (ONT) MinION device offers the same advantages as other NGS platforms but is also portable and inexpensive, allowing in-field deployment on a larger scale. ONTbased sequencing was shown suitable for the surveillance and identification of viral and bacterial pathogens that infect humans or plants [21][22][23][24], and can distinguish between single-nucleotide variants to resolve different isolates, strains, and subspecies [25][26][27].…”

Section: Introductionmentioning

confidence: 99%

Real-Time On-Site Diagnosis of Quarantine Pathogens in Plant Tissues by Nanopore-Based Sequencing

et al. 2022

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Rapid and sensitive assays for the identification of plant pathogens are necessary for the effective management of crop diseases. The main limitation of current diagnostic testing is the inability to combine broad and sensitive pathogen detection with the identification of key strains, pathovars, and subspecies. Such discrimination is necessary for quarantine pathogens, whose management is strictly dependent on genotype identification. To address these needs, we have established and evaluated a novel all-in-one diagnostic assay based on nanopore sequencing for the detection and simultaneous characterization of quarantine pathogens, using Xylella fastidiosa as a case study. The assay proved to be at least as sensitive as standard diagnostic tests and the quantitative results agreed closely with qPCR-based analysis. The same sequencing results also allowed discrimination between subspecies when present either individually or in combination. Pathogen detection and typing were achieved within 13 min of sequencing owing to the use of an internal control that allowed to stop sequencing when sufficient data had accumulated. These advantages, combined with the use of portable equipment, will facilitate the development of next-generation diagnostic assays for the efficient monitoring of other plant pathogens.

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Nanopore sequencing for the detection and identification of Xylella fastidiosa subspecies and sequence types from naturally infected plant material

Cited by 19 publications

References 43 publications

Investigating plant disease outbreaks with long-read metagenomics: sensitive detection and highly resolved phylogenetic reconstruction applied to Xylella fastidiosa

Investigating plant disease outbreaks with long-read metagenomics: sensitive detection and highly resolved phylogenetic reconstruction applied to Xylella fastidiosa

Metagenomic Sequencing for Identification of Xylella fastidiosa from Leaf Samples

Real-Time On-Site Diagnosis of Quarantine Pathogens in Plant Tissues by Nanopore-Based Sequencing

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